Approximately one third of the genes in the human genome encode secreted and plasma membrane proteins that are synthesized on membrane-bound ribosomes at the endoplasmic reticulum (ER). These proteins cross or become embedded in the ER membrane as they are being synthesized, i.e., co-translationally. The ribosomes synthesizing this subclass of proteins are brought to the ER membrane by an evolutionarily conserved molecular machine, the signal recognition particle (SRP), and its ER membrane-embedded receptor. The folding of the growing polypeptide chain is assisted co-translationally by the sequential engagement of different chaperones and disulfide isomerases in the ER, where the protein folding status is constantly monitored. In response to a folding imbalance, or ER stress, a network of signaling pathways, collectively called the unfolded protein response (UPR) readjusts the ER's protein folding capacity to accommodate the load of proteins entering its lumen. The UPR sensors in the ER membrane respond to an accumulation of un- or misfolded proteins in the ER and initiate appropriate preemptive (i.e. fewer clients are allowed to enter the ER) or corrective (i.e. the ER processing capacity is enlarged) actions. Of the UPR sensors found in mammalian cells, IRE1 is the most evolutionarily conserved and best understood. It is a bifunctional ER-resident transmembrane kinase/RNase that upon activation excises an intron from the mRNA encoding its principal effector, the transcription factor XBP1. This induces a frameshift in the XBP1 open reading frame, and leads to production of functional XBP1 transcription factor that initiates a genetic program to adjust the ER's protein folding capacity according to need. To date, the sensing of the ER's folding conditions and the resulting IRE1-mediated signal transduction events have been thought of as autonomous events, occurring after and uncoupled from protein synthesis and translocation. This view has changed profoundly with two independent and complementary discoveries supporting the synchronous monitoring of the protein folding status in the ER by IRE1 and co-translational translocation of ER clients into its lumen. Using RNA crosslinking techniques coupled with deep sequencing, we sought to comprehensively catalog mRNA substrates for IRE1. To our surprise, we found that major crosslinks between IRE1 and RNA mapped to the SRP RNA and ribosomal RNA. Moreover, the crosslink sites converge on a functionally well-defined region on the ribosome (to SRP RNA's Alu domain and to rRNA in proximity of where SRP's Alu domain binds). In addition, we have shown directly that the purified cytosolic portion of IRE1 binds with high affinity to ribosomes. The notion that IRE1 may function on translating ribosomes is underscored by the independent discoveries of Plumb et al. (eLife 2015), who identified a contact between IRE1's lumenal domain and the Sec61 translocon. These investigators mapped and probed the functional importance of this interaction by mutational analyses and showed impairment in IRE1 function. Taken together, these results suggest that IRE1 can identify and cleave substrates co-translationally. In this proposal, we will focus on deepening our understanding of the IRE1-ribosome-SRP interactions, aiming to add solid structural and mechanistic understanding to lay the foundations that then can allow us to test the importance of these newly characterized interactions in vivo. To this end, we will (i) determine IRE1's engagement with different stages of the targeting/translocation process, (ii) isolate IRE1-engaged ribosomes from living cells and characterize their composition and translational status, (iii) determine the structure of IRE1 in complex with the co-translational targeting machinery by cryo- EM, and (iv) determine the functional significance of the IRE1-SRP and IRE1-ribosome interactions by complementary approaches employing both biochemical assays and in vivo mutagenesis approaches. In this way, we seek to build a conceptually new toolbox, which we expect will lead us to expand and refine-if not substantially revise-current models of IRE1 engagement in mammalian cells.